Spider Silk-production and Biomedical Applications - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 150

hexafluoroisopropanol (HFIP) or formic acid (Agapov et al., 2009; Arcidiacono et al.,
1998; Arcidiacono et al., 2002; Bini et al., 2006; Bogush et al., 2008; Exler et al.,
2007; Fahnestock and Bedzyk, 1997; Fahnestock and Irwin, 1997; Fukushima, 1998;
Geisler et al., 2008; Huemmerich et al., 2004a; Huemmerich et al., 2006; Junghans,
2006; Lammel, 2008; Lazaris et al., 2002; Lewis et al., 1996; Liebmann, 2008; Lin
et al., 2009; Mello et al., 2004; Rammensee et al., 2008; Scheller et al., 2001; Slotta,
2006; Slotta et al., 2008; Teulé et al., 2009; Wong Po Foo et al., 2006; Zhou et al.,
2001). After removal of the solvents used for solubilisation, it has generally been dif-
ficult to control assembly of the denatured spidroins into solid structures. The spider
can store the spidroins in an aqueous solution and precisely regulate the conversion
into fibers during spinning. Several strategies have been tried in order to mimic this
process; to first prevent and then promote assembly and thereby avoid resolubilisa-
tion steps. For example introduction of methionine residues for controlled oxidation/
reduction ( Szela et al., 2000; Valluzzi et al., 1999; Winkler et al., 1999) have been
utilised. A kinase recognition motif has also been used for controlled assembly via
enzymatic phosphorylation (Winkler et al., 2000). When fused to the solubility tag
thioredoxin a miniature spidroin composed of four poly-Ala/Gly-rich co-segments
and the C-terminal domain, 4RepCT, has been successfully produced in soluble form
(Stark et al., 2007). Upon proteolytic release from the solubilising thioredoxin partner
the miniature spidroin 4RepCT spontaneously forms macroscopic fibers that resemble
native spider silk (Stark et al., 2007). Together with minispidroins recombinantly pro-
duced in the cytosol of insect cells (Huemmerich et al., 2004b) this is so far the only
report of spontaneous self assembly into silk-like fibers, indicating the importance of
non-denaturing conditions during production.
Processing into solid spidroin structures
Several different methods to convert denatured recombinant spidroins into fibers
have been used, example wet spinning (Arcidiacono et al., 2002; Bogush et al., 2008;
Brooks et al., 2008; Lazaris et al., 2002; Lewis et al., 1996; Teulé et al., 2007; Teulé et
al., 2009; Yang et al., 2005), hand drawing (Exler et al., 2007; Teulé et al., 2007), spin-
ning though microfluidic devices (Rammensee et al., 2008) and electrospinning ( Bini
et al., 2006; Bogush et al., 2008; Stephens et al., 2005; Wong Po Foo et al., 2006; Zhou
et al., 2008). Other formats than fibers are preferred for some applications, example
matrices for cell culture and tissue engineering. Therefore, materials have been pro-
cessed into films (Arcidiacono et al., 2002; Bini et al., 2006; Fukushima, 1998; Huang
et al., 2007; Huemmerich et al., 2006; Junghans, 2006; Metwalli, 2007 Scheller et al.,
2004; Slotta, 2006; Szela et al., 2000; Valluzzi et al., 1999; Winkler et al., 1999; Wong
Po Foo et al., 2006), microbeads (Liebmann, 2008), microspheres (Lammel, 2008;
Slotta et al., 2008), and microcapsules (Hermanson et al., 2007). Three-dimensional
porous scaffolds have also been produced by salt leaching methods (Agapov et al.,
2009). Most of these techniques require post treatment in dehydrating or salting-out
solvents, to increase the β-sheet contents and stability. In contrast, the recombinant
miniature spidroin 4RepCT can be used to produce fibers directly in physiological
buffers (Figure 1). The self-assembled fibers form bundles of varying thickness with a
surface with lengthwise pattern of irregular grooves and ridges. The 4RepCT can also